Most of our PET-CT studies to date have used 18F-2-fluoro-2-deoxyglucose (FDG) to image the metabolism of the eukaryotic cells in TB lesions but we are also making attempts to identify the location, abundance and metabolic state of the bacteria in lesions. In an effort to identify small molecules that could be used to specifically label MTb in vivo, we capitalized on the unusually broad substrate tolerance of the MTb antigen 85 enzymes, which transfer mycolates onto structurally diverse sugars to form part of MTbs cell wall. Antigen-85 enzymes are expressed on the exterior of MTbs cell wall and incorporate exogenous trehalose (a nonmammalian disaccharide consisting of a two 1-1 , -linked glucose monomers) as either the mono- or dimycolate, even tolerating trehalose molecules containing bulky modifications. We have used this system to chemically incorporate 18F trehalose into bacteria in the lesions of infected rabbits. Use of trehalose is unique to bacteria, which should limit noise in PET scans. The Davis group has designed three trehalose analogs incorporating fluorine at the 2, epi-4, or 6-position of trehalose for use as PET radiotracers. The 2-fluorotrehalose (FDT) synthesis is a biomimetic process, inspired by bacterial synthesis of trehalose from glucose. Chemoenzymatic synthesis of FDT occurs as a one-pot cascade reaction in which hexokinase transfers a phosphate from adenosine triphosphate (ATP) to FDG (normally glucose in bacterial trehalose synthesis). OtsA then transfers the glucose from the donor UDP-glucose to the acceptor phosphorylated FDG. Dephosphorylation to give the desired product is effected by OtsB. The advantage here is that a relatively technically facile manipulation would convert a commercially available radiotracer to a TB-specific one. We have acquired some preliminary PET-CT scan data in rabbits using the FDT probe as well, one healthy, three infected with HN878 MTb. In the first infected animal, four lesions were present. Two were not PET-active, and two were, although all four had similar amounts of colony forming units (CFU). Upon necropsy, the two PET-inactive lesions were extremely rigid and thick-walled, implying that uptake is related more to accessibility (i.e. vasculature) than amount of bacteria present. Intriguingly, the second two rabbits showed more complex lesions, which clearly displayed differential labeling between the 18F-FDG and FDT. Metabolism data was inconclusive but suggested some metabolism (as much as 20%) of the probe from FDT back info FDG. It is assumed this happens via trehalase natively expressed by the rabbits. We then evaluated FDT in Mtb-infected marmosets and macaques, as these animals should express lower levels of trehalase and are a more physiologically relevant model to human disease. The marmosets and macaques showed no metabolism of the FDT back to FDG. The FDT continued to show differential labeling compared to FDG. In one marmoset, we treated with the first-line regimen of izoniazid, pyrazinamide, rifampicin, and ethambutol for five weeks, with dosing five days a week. The animal showed very low disease burden upon necropsy as judged by CFU. The FDT PET-CT scans reflected this low burden, showing a clear reduction in uptake compared to pre-treatment scans. The FDG did not show a significant decrease in uptake and in some lesions displayed increase uptake. This is an extremely promising sign that the FDT will be able to give an earlier indication of treatment success or failure as compared to FDG. We have performed biodistribution studies by analyzing tissue samples from relevant organs in two marmosets, which showed little uptake in uninfected tissue other than kidneys, the route for clearance of the radiotracer. We have recently successfully performed a biodistribution study via PET-CT in 3 naive macaques to provide data for first in human dosing studies. We have optimized the current 1 hour reaction protocol, the enzyme assays have been established to characterize both enzymes, OtsA and OtsB, during the chemoenzymatic synthesis of 2-deoxy-2-18FFluoro-trehalose. The calculated specific activity for OtsB is that one unit of OtsB can convert 1.0 micromole of trehalose-6-phosphate to 1.0 micromole of trehalose per minute at pH 7.5 at 37 C. The activity of OtsA was determined by monitoring the production of trehalose-6-phosphate, afterwards, to quantify the amount of trehalose-6-phosphate in each reaction sample, the reaction mixture was incubated for 30 min at 37C with 1 g OtsB. After the OtsB reaction, the amount of trehalose produced was determined using a commercial assay and the data were converted to the amount of trehalose-6-phosphate since a molecule of trehalose-6-phosphate is used to produce a molecule of trehalose. For the specific activity of OtsA, one unit of OtsA can convert 1.0 micromole of glucose-6-phosphate to 1.0 micromole of trehalose-6-phosphate per minute at pH 7.5 at 37 C. Based on the calculated specific activity of both OtsA and OtsB, it turned out that the current reaction protocol can be optimized. The production rate prediction study using the specific activity of the enzymes, followed by a small scale one-pot synthesis, showed that the reaction time can be decreased in the current reaction condition. The addition of 40 ul 5M NaCl to the reaction could enhance the protein stability during the reaction, providing more reliable and consistent reaction condition. Overall, these data suggested that the reaction time can be shortened to 30 min and the feasibility of the time reduction will need to be tested in the actual probe synthesis. Since the quantification of 2-deoxy-2-18FFluoro-trehalose is required to have FDA-approval for the clinical study to test 18FFDT as a PET scan probe, the enzymatic assay to quantify the amount of 2-deoxy-2-18FFluoro-trehalose has been established. The active ingredient 2-deoxy-2-18FFluoro-trehalose has a molecular formula of C12H2118FO10 with a molecular weight of 343.29 daltons. After a certain period of time, 2-deoxy-2-18FFluoro-trehalose decays into 2-18O-trehalose. The amount of the cold starting material, 18OGlucose, and the final product, 18Otrehalose, has been detected and analyzed. In commercially available FDG sample, the average amount of FDG was 90 ug in 600 ul sample and the calculated average specific activity was 66 Ci/mmol. In enzymatically synthesized FDG samples using the 600 ul FDG described above, the average amount of FDT was 99 ug and the specific activity was 29.3 Ci/mmol. With this quantification result, the toxicology study on this molecule has been designed and we are waiting on the production of the cold 19FFTD for use in the rat and dog toxicology studies. Well seek funding for these studies once it is certain that the cold FTD has similar impurities as are observed in the enzymatically synthesized 18FFDT. A draft human protocol has also been composed and reviewed by collaborators in the clinical center and in Cape Town, South Africa where the large study will be positioned.